PROJECT SUMMARY Staphylococcus aureus is a ubiquitous human pathogen, resulting in superficial, invasive, and disseminated infections. One of the most common invasive manifestations of S. aureus disease is osteomyelitis, a frequently occurring and debilitating infection of bone. Osteomyelitis triggers dramatic alterations in bone architecture, leading to severe complications such as bone destruction, pathologic fractures, and growth defects. An emerging body of literature suggests that both local and systemic inflammation trigger altered interactions between bone-forming osteoblasts and bone-resorbing osteoclasts to impact bone homeostasis. Skeletal cells are known to express innate pattern recognition receptors (PRRs), but the contribution of innate sensing towards bone homeostasis and antibacterial immunity during S. aureus osteomyelitis has not yet been explored. The overarching objective of this proposal is to characterize how innate sensing of bacterial pathogens by skeletal cells triggers alterations in bone physiology. In order to define the impact of skeletal cell PRRs on bone homeostasis, we first focused on the critical signaling adaptor protein, MyD88, which is necessary to transduce signals through toll-like receptors (TLRs) and IL-1 receptors (IL-1R). Our preliminary data demonstrate that MyD88 is necessary to control S. aureus replication and dissemination in vivo and that osteoclast differentiation can be stimulated by bacterial components in a MyD88-dependent manner in vitro. Therefore, the central hypothesis of this proposal is that S. aureus modulates osteoclast precursor cell biology and bone remodeling through ligation of osteoclast PRRs and the induction of inflammation. To test this hypothesis, I will use a newly developed murine S. aureus osteomyelitis model from our laboratory. This model is advantageous compared to other osteomyelitis models because it allows us to utilize genetically modified animals, high-resolution quantitative imaging analysis, and unique histologic techniques for quantifying perturbations in bone remodeling. Experiments proposed in Aim 1 will investigate the roles of TLR and IL-1R signaling on osteoclast differentiation by monitoring osteoclastogenic signaling cascades, transcription factor activity, expression of mature osteoclast markers, and functionality of osteoclasts formed in vitro. Aim 2 will explore how MyD88 signaling in skeletal cells impacts clearance of S. aureus and bone remodeling. Collectively, these data will define signaling crosstalk between canonical osteoclast differentiation and innate immune pathways to activate osteoclast differentiation and maturation programs. Additionally, these findings will describe how MyD88 signaling in skeletal cells contributes to immune defenses and affects the kinetics of bone remodeling. This proposed work will have broad implications for how innate skeletal cell sensing contributes to the development of an effective immune response and influences bone homeostasis.